scholarly journals Involvement of the Zinc-Binding Capacity of Sendai Virus V Protein in Viral Pathogenesis

2000 ◽  
Vol 74 (17) ◽  
pp. 7834-7841 ◽  
Author(s):  
Cheng Huang ◽  
Katsuhiro Kiyotani ◽  
Yutaka Fujii ◽  
Noriko Fukuhara ◽  
Atsushi Kato ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Sendai Virus ◽  
Binding Capacity ◽  
Viral Pathogenesis ◽  
Growth Patterns ◽  
Zinc Binding ◽  
Wild Type ◽  
V Protein ◽  
Rich Domain ◽  
Cysteine Rich Domain

ABSTRACT The V protein of Sendai virus (SeV) is nonessential to virus replication in cell culture but indispensable to viral pathogenicity in mice. The highly conserved cysteine-rich zinc finger-like domain in its carboxyl terminus is believed to be responsible for this viral pathogenicity. In the present study, we showed that the cysteine-rich domain of the SeV V protein could actually bind zinc by using glutathione-S-transferase fusion proteins. When the seven conserved cysteine residues at positions 337, 341, 353, 355, 358, 362, and 365 were replaced individually, the zinc-binding capacities of the mutant proteins were greatly impaired, ranging from 22 to 68% of that of the wild type. We then recovered two mutant SeVs from cDNA, which have V-C341S and V-C365R mutations and represent maximal and minimal zinc-binding capacities among the corresponding mutant fusion proteins, respectively. The mutant viruses showed viral protein synthesis and growth patterns similar to those of wild-type SeV in cultured cells. However, the mutant viruses were strongly attenuated in mice in a way similar to that of SeV VΔC, which has a truncated V protein lacking the cysteine-rich domain, by exhibiting earlier viral clearance from the mouse lung and less virulence to mice. We therefore conclude that the zinc-binding capacity of the V protein is involved in viral pathogenesis.

scholarly journals Sendai Virus Targets Inflammatory Responses, as Well as the Interferon-Induced Antiviral State, in a Multifaceted Manner

2003 ◽  
Vol 77 (14) ◽  
pp. 7903-7913 ◽  
Author(s):  
Laura Strähle ◽  
Dominique Garcin ◽  
Philippe Le Mercier ◽  
Joerg F. Schlaak ◽  
Daniel Kolakofsky
Keyword(s):  
Immune Responses ◽  
Inflammatory Responses ◽  
Sendai Virus ◽  
Wild Type ◽  
C Protein ◽  
Multifaceted Approach ◽  
V Protein ◽  
Antiviral State ◽  
Adaptive Immune ◽  
Cellular Genes

ABSTRACT We have used cDNA arrays to compare the activation of various cellular genes in response to infection with Sendai viruses (SeV) that contain specific mutations. Three groups of cellular genes activated by mutant SeV infection, but not by wild-type SeV, were identified in this way. While some of these genes are well known interferon (IFN)-stimulated genes, others, such as those for interleukin-6 (IL-6) and IL-8, are not directly induced by IFN. The gene for beta IFN (IFN-β), which is critical for initiating an antiviral response, was also specifically activated in mutant SeV infections. The SeV-induced activation of IFN-β was found to depend on IFN regulatory factor 3, and the activation of all three cellular genes was independent of IFN signaling. Mutations that disrupt four distinct elements in the SeV genome (the leader RNA, two regions of the C protein, and the V protein) all lead to enhanced levels of IFN-β mRNA, and at least three of these viral genes also appear to be involved in preventing activation of IL-8. Our results suggest that SeV targets the inflammatory and adaptive immune responses as well as the IFN-induced intracellular antiviral state by using a multifaceted approach.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Kras Mutations ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

AbstractThe first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we present crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures reveal that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutations at the KRAS-CRD interface result in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

Siamese cat tyrosinase has enhanced proteasome degradation and increased cellular aggregation

2020 ◽  
Author(s):  
Ingrid R. Niesman
Keyword(s):  
Proteasome Inhibition ◽  
Cellular Biology ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Rich Domain ◽  
Increased Risk ◽  
Siamese Cat ◽  
Cellular Aggregation ◽  
Tyrosinase Enzyme ◽  
Cysteine Rich Domain

AbstractSiamese cats are a notable example of a temperature-sensitive partial albinism phenotype. The signature color-pointing pattern is the result of an amino acid substitution – G302R – in the cysteine-rich domain of feline tyrosinase. The precise mechanism for the loss of tyrosinase enzyme activity due to this mutation is unknown.ObjectiveWe have used a cellular biology approach to begin unravel relationships between feline coloration, behavior and increased risk for feline cognitive dysfunction syndrome. GFP-fusion constructs of wild type domestic short hair tyrosinase and Siamese (G302R) tyrosinase generated to study cellular trafficking, degradation and the propensity for cellular aggregation.Data DescriptionC-terminal GFP G302R expression has reduced Golgi localization, increased cytosolic fractions with reduced calnexin co-localization. N-terminal GFP constructs were retained in the ER, with little to no Golgi associated forms. C-terminal and N-terminal GFP G302R TYR is observed to have increased high molecular weight aggregation following proteasome inhibition.

Zinc co-ordination by the DHHC cysteine-rich domain of the palmitoyltransferase Swf1

2013 ◽  
Vol 454 (3) ◽  
pp. 427-435 ◽  
Author(s):  
Ayelén González Montoro ◽  
Rodrigo Quiroga ◽  
Javier Valdez Taubas
Keyword(s):  
Amino Acids ◽  
Tertiary Structure ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Essential Amino Acids ◽  
Zinc Binding ◽  
Loss Of Function ◽  
Post Translational Modification ◽  
Rich Domain ◽  
Cysteine Rich Domain

S-acylation, commonly known as palmitoylation, is a widespread post-translational modification of proteins that consists of the thioesterification of one or more cysteine residues with fatty acids. This modification is catalysed by a family of PATs (palmitoyltransferases), characterized by the presence of a 50-residue long DHHC-CRD (Asp-His-His-Cys cysteine-rich domain). To gain knowledge on the structure–function relationships of these proteins, we carried out a random-mutagenesis assay designed to uncover essential amino acids in Swf1, the yeast PAT responsible for the palmitoylation of SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins. We identified 21 novel loss-of-function mutations, which are mostly localized within the DHHC-CRD. Modelling of the tertiary structure of the Swf1 DHHC domain suggests that it could fold as a zinc-finger domain, co-ordinating two zinc atoms in a CCHC arrangement. All residues predicted to be involved in the co-ordination of zinc were found to be essential for Swf1 function in the screen. Moreover, these mutations result in unstable proteins, in agreement with a structural role for these zinc fingers. The conservation of amino acids predicted to form each zinc-binding pocket suggests a shared function, as the selective pressure to maintain them is lost upon mutation of one of them. A Swf1 orthologue that lacks one of the zinc-binding pockets is able to complement a yeast swf1∆ strain, possibly because a similar fold can be stabilized by hydrogen bonds instead of zinc co-ordination. Finally, we show directly that recombinant Swf1 DHHC-CRD is able to bind zinc. Sequence analyses of DHHC domains allowed us to present models of the zinc-binding properties for all PATs.

scholarly journals KRAS interaction with RAF1 RAS-binding domain and cysteine-rich domain provides insights into RAS-mediated RAF activation

2020 ◽  
Author(s):  
Timothy H. Tran ◽  
Albert H. Chan ◽  
Lucy C. Young ◽  
Lakshman Bindu ◽  
Chris Neale ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Crystal Structures ◽  
Kras Mutation ◽  
Regulatory Region ◽  
Binding Domain ◽  
Published Data ◽  
Wild Type ◽  
Rich Domain ◽  
Structural Entity ◽  
Cysteine Rich Domain

ABSTRACTA vital first step of RAF activation involves binding to active RAS, resulting in the recruitment of RAF to the plasma membrane. To understand the molecular details of RAS-RAF interaction, we solved crystal structures of wild-type and oncogenic mutants of KRAS complexed with the RAS-binding domain (RBD) and the membrane-interacting cysteine-rich domain (CRD) from the N-terminal regulatory region of RAF1. Our structures revealed that RBD and CRD interact with each other to form one structural entity in which both RBD and CRD interact extensively with KRAS. Mutation at the KRAS-CRD interface resulted in a significant reduction in RAF1 activation despite only a modest decrease in binding affinity. Combining our structures and published data, we provide a model of RAS-RAF complexation at the membrane, and molecular insights into RAS-RAF interaction during the process of RAS-mediated RAF activation.

scholarly journals The first cysteine-rich domain of the receptor GFRα1 stabilizes the binding of GDNF

2005 ◽  
Vol 387 (3) ◽  
pp. 817-824 ◽  
Author(s):  
Heidi VIRTANEN ◽  
Jianmin YANG ◽  
Maxim M. BESPALOV ◽  
Jukka O. HILTUNEN ◽  
Veli-Matti LEPPÄNEN ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Full Length ◽  
Tyrosine Kinase Receptor ◽  
Soluble Receptors ◽  
Truncated Receptor ◽  
Rich Domain ◽  
Low Concentrations ◽  
Optimal Function ◽  
High Concentrations ◽  
Cysteine Rich Domain

The GDNF (glial cell line-derived neurotrophic factor)-binding receptor GFRα1 (GDNF family receptor α1) is attached to the membrane by a GPI (glycosylphosphatidylinositol) anchor and consists of three cysteine-rich domains. The region corresponding to the second and third domains has been shown previously to participate in ligand binding, and to interact with the transmembrane tyrosine kinase receptor RET. No function has so far been found for the N-terminal, first domain (D1). Here we show that the GPI-anchored full-length receptor binds 125I-GDNF two times more tightly than does a GPI-anchored truncated receptor lacking D1. Scintillation proximity assays with purified receptor proteins also show that the GDNF-binding capacity of the soluble full-length GFRα1 is two times higher than the GDNF-binding capacity of the soluble D1-truncated GFRα1. As RET stabilizes the binding of GDNF equally well to the full-length and truncated receptors, D1 seems not to be involved in the interaction between GFRα1 and RET. Moreover, soluble full-length GFRα1 mediates GDNF-promoted neurite outgrowth in PC6-3 cells more efficiently than the soluble truncated GFRα1 protein. At low concentrations, the soluble fulllength receptor mediates the phosphorylation of RET more efficiently than the soluble truncated receptor. However, when the receptors are overexpressed on the cell surface as GPI-anchored proteins, or added to the growth medium at high concentrations as soluble proteins, full-length and truncated GFRα1 are indistinguishable in GDNF-dependent RET-phosphorylation assays. High levels of the receptors can thus mask a slightly impaired function in the phosphorylation assay. Based on assays with both GPI-anchored and soluble receptors, we therefore conclude that D1 contributes to the optimal function of GFRα1 by stabilizing the interaction between GFRα1 and GDNF.

ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3232-3237 ◽  
Author(s):  
Kenji Soejima ◽  
Masanori Matsumoto ◽  
Koichi Kokame ◽  
Hideo Yagi ◽  
Hiromichi Ishizashi ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Single Point ◽  
Wild Type ◽  
Functional Relationships ◽  
Rich Domain ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Remarkable Reduction ◽  
Cysteine Rich Domain

AbstractA severe lack of von Willebrand factor–cleaving protease (VWF-CP) activity can cause thrombotic thrombocytopenic purpura (TTP). This protease was recently identified as a member of the ADAMTS family, ADAMTS-13. It consists of a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, additional Tsp1 repeats, and CUB domains. To explore the structural and functional relationships of ADAMTS-13, we prepared here 13 sequential COOH-terminal truncated mutants and a single-point mutant (ArgGlyAsp [RGD] to ArgGlyGlu [RGE] in the cysteine-rich domain) and compared the activity of each mutant with that of the wild-type protein. The results revealed that the truncation of the cysteine-rich/spacer domains caused a remarkable reduction in VWF-CP activity. We also prepared immunoglobulin G (IgG) fractions containing inhibitory autoantibodies against ADAMTS-13 from plasma from 3 patients with acquired TTP, and we performed mapping of their epitopes using the aforementioned mutants. The major epitopes of these antibodies were found to reside within the cysteine-rich/spacer domains. These results suggest that the ADAMTS-13 cysteine-rich/spacer domains are essential for VWF-CP activity.

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